EP1274466A1 - Composition de detection de beta-1,3-glucane, procede de preparation de cette derniere et trousse de diagnostic permettant de detecter le beta-1,3-glucane - Google Patents

Composition de detection de beta-1,3-glucane, procede de preparation de cette derniere et trousse de diagnostic permettant de detecter le beta-1,3-glucane

Info

Publication number
EP1274466A1
EP1274466A1 EP01942566A EP01942566A EP1274466A1 EP 1274466 A1 EP1274466 A1 EP 1274466A1 EP 01942566 A EP01942566 A EP 01942566A EP 01942566 A EP01942566 A EP 01942566A EP 1274466 A1 EP1274466 A1 EP 1274466A1
Authority
EP
European Patent Office
Prior art keywords
glucan
composition
calcium ions
phenoloxidase
fractions
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP01942566A
Other languages
German (de)
English (en)
Other versions
EP1274466A4 (fr
Inventor
Joong Hyuck Auh
Bu Soo Park
Chang Hun Joo
Chong Jin Park
Bok Luel Lee
Kum Young Lee
Seung-Suh Hong
Hyun-Soo Lee
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Samyang Genex Corp
Original Assignee
Samyang Genex Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Samyang Genex Corp filed Critical Samyang Genex Corp
Publication of EP1274466A1 publication Critical patent/EP1274466A1/fr
Publication of EP1274466A4 publication Critical patent/EP1274466A4/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase

Definitions

  • the present invention relates to a composition for detecting ⁇ -1 ,3-glucan, a
  • Phenoloxidase acting in this process exists in the insect's body as a prophenoloxidase in ordinary times, and the prophenoloxidase is converted to phenoloxidase as the final activated form by the stimulus of the final product of the prophenoloxidase chain reaction.
  • lipopolysaccha de, peptidoglycan and other cell wall components of the microorganisms lipopolysaccha de, peptidoglycan and other cell wall components of the microorganisms.
  • Prophenoloxidase exists in the body of holometabola and is activated to
  • composition capable of reacting specifically with ⁇ -1 ,3-glucan
  • US patent 5,266,461 discloses a reagent for determining ⁇ -1 ,3-glucan
  • the present invention relates to a composition for detecting ⁇ -1 ,3-glucan.
  • the present invention further relates to a preparation method of a composition for detecting ⁇ -1 ,3-glucan.
  • the present invention also relates to a method of detecting ⁇ -1 ,3-glucan.
  • the present invention still further relates to a diagnostic kit for detecting
  • the present invention relates to a composition for detecting ⁇ -1 ,3-glucan
  • phenoloxidase system refers to a system that
  • phenoloxidase composition refers to a composition which comprises all or some components of the phenoloxidase
  • the present invention relates to a composition that shows phenoloxidase
  • composition in the present invention includes all or some components of the phenoloxidase system in insects and includes prophenoloxidase, for example.
  • the present invention relates to a composition for detecting ⁇ -1 , 3-
  • glucan preferably the minimum down to 20 pg/ml.
  • composition for detecting ⁇ -1 ,3-glucan of the present invention can be any composition for detecting ⁇ -1 ,3-glucan of the present invention.
  • an insect or “insects” refers to an insect or insects that has a phenoloxidase system in the body, and preferably a holometabola insect.
  • the examples of insect include those belonging to
  • Crustaceans such as lobsters and shrimps and Coleoptera, and more preferably Tenebrionidae and Scarabaeidae orders of Coleoptera.
  • the present invention relates to a preparation method of a
  • composition exhibiting phenoloxidase activity by ⁇ -1 ,3-glucan in the presence of
  • the method in the present invention includes a step to obtain a sample comprising a mixture of plasma and hemocyte lysate from insects, a step to obtain fractions by treating the sample with a solvent or buffer solution containing a sufficient amount of a chelating agent that can chelate the calcium ions existing in the sample and in the present separation process, and a step to
  • Another example of the method of the present invention includes a step to obtain plasma from insects, a step to obtain fractions by treating the plasma with a solvent or buffer solution containing a sufficient amount of a chelating agent that can chelate the calcium ions existing in the plasma and in the present separation process, a step to add the hemocyte lysate or partially purified hemocyte lysate to said fractions, and a step to select the fractions exhibiting phenoloxidase activity by ⁇ -1 ,3-glucan in the presence of calcium ions.
  • the present invention provides a method for preparing
  • the method of preparing a phenoloxidase composition according to the present invention includes obtaining a sample comprising hemolymph or a mixture of plasma and hemocyte lysate in the presence of sufficient amount of chelating agent that can chelate the calcium ions possibly existing in the above sample and in the present separation process to obtain the phenoloxidase composition, obtaining fractions by treating the sample with a solvent or buffer solution containing a sufficient amount of the chelating agent that can chelate the calcium ions existing in the above sample and during the separation process,
  • an anticoagulant buffer solution that can prevent the coagulation of the hemolymph is preferably used while
  • hemolymph more preferably a citrate buffer solution
  • composition which exhibits the phenoloxidase activity specifically by ⁇ -1 ,3-
  • glucan in the presence of calcium ions can be obtained by using a mixture of
  • composition of the present invention is insect's plasma and hemocytes.
  • hemocytes can be obtained by collecting the hemolymph of insects, separating
  • hemocytes plasma and hemocytes is adding hemocyte lysate or partially purified hemocyte
  • the mixture of insect's plasma and hemocytes can be obtained from hemolymph
  • hemolymph or separated hemocytes can be
  • chelating agent can be performed by column chromatography, for example
  • any of chelating agents known in the field can be used without limitation, and can be EDTA, EGTA or citric acid, for example.
  • the amount of the chelating agent can be various depending on the conditions of the separation process such as the sample, the kind of column and the kind of solvent, and needs to enough to chelate the calcium ions in the insect sample and during the separation process. Therefore, those of ordinary skill in the field would be able to easily determine the amount of the chelating agent without undue experimentation.
  • the solvent or buffer solution is not limited in the preparation method according to the present invention, but is preferably that of not more than pH 6.5.
  • pH is higher than 6.5
  • serine protease a component involved in the phenoloxidase cascade reaction, can be activated and causes activation of prophenoloxidase into phenoloxidase. It results to make it difficult to obtain the composition according to the present invention.
  • One of the examples to treat the insect sample with the solvent or buffer solution containing a chelating agent is by column chromatography.
  • a column packed with resin can be loaded with an insect sample, and eluted with the solvent or buffer solution containing a chelating agent to obtain fractions.
  • composition that can detect ⁇ -1 ,3-glucan specifically down the composition that can detect ⁇ -1 ,3-glucan specifically down
  • the resin that can be used for column chromatography in the present invention is preferably the resin that uses dextran or vinyl as a raw material.
  • dextran or vinyl for example, Sephadex or Toyopearl can be used.
  • composition according to the present invention can be used in any combination.
  • the present invention relates to a method of diagnosing
  • invention includes the steps of collecting a sample from a specimen, adding
  • composition of the sample for example, the composition of the sample
  • the above insect plasma and hemocyte lysate can be
  • the specimen to be tested may be animals including humans or any combination thereof
  • the fungal infection can be diagnosed by:
  • microorganisms like fungi, that have ⁇ -1 ,3-glucan as a cell well component in the cultivating industry.
  • microorganisms that have ⁇ -1 ,3-glucan as a cell wall component, pretreatment
  • lipopolysaccharide that may exist in a specimen sample
  • the influence of lipopolysaccharide can be eliminated by treating the specimen sample with materials such as polymyxin that can bind specifically with or precipitate lipopolysaccharide.
  • any determination method of phenoloxidase activity known in the field can be used in its present condition or with some modifications as the method of determining phenoloxidase activity that can be used as a diagnostic method of fungal infection in the present invention.
  • the phenoloxidase activity can be determined easily at the early stage of the fungal infection by measuring the absorbance of the color developed by the 4-methylcatechol/4- hydroxyproline ethyl ester (4-MC/4-HP) or from the melanin formation reaction using dopamine.
  • the present invention relates to a diagnostic kit for detecting ⁇ -1 ,3-
  • the diagnostic kit for detecting ⁇ -1 ,3-glucan contains the composition
  • composition that exhibits the phenoloxidase exhibits the phenoloxidase
  • a sample comprising plasma or a mixture of plasma and hemocyte lysate from an insect; treating said sample with a solvent or buffer solution containing a sufficient amount of a chelating agent to chelate calcium ions existing in said sample and in the present separation process to obtain fractions therefrom; and selecting fractions exhibiting phenoloxidase activity by
  • the buffer solution and the method to determine the phenoloxidase activity used in the present invention are as follows.
  • Anticoagulation buffer solution pH 5.5: NaCI 15 mM, trisodium citrate
  • ⁇ -1 ,3-glucan solution a solution prepared by mixing 10 ⁇ l of the solution
  • composition was added and pre-reacted for 10 min at 30 °C. After adding 405 ⁇ l of 20 mM Tris buffer solution (pH 8.0), 5 ⁇ l of 1 M CaCI 2 and 50 ⁇ l of 10 mM
  • the amount of ⁇ -1 ,3-glucan in the sample can be determined from the
  • Figure 1 is a graph showing the phenoloxidase activity of the phenoloxidase composition isolated from the hemolymph of Tenebrio molitor
  • Figure 2 is a standard curve of the phenoloxidase activity of the phenoloxidase composition isolated from the hemolymph of Tenebrio molitor
  • Figure 3 is a graph examining the detection specificity in relative to lipopolysaccharide of the phenoloxidase composition isolated from the hemolymph of Tenebrio molitor,
  • Figure 4 is a graph examining the detection specificity in relative to peptidoglycan of the phenoloxidase composition isolated from the hemolymph of Tenebrio molitor
  • Figure 5 is a graph comparing the phenoloxidase activity induced by ⁇ - 1 ,3-glucan in each composition obtained from plasma, hemocyte lysate and hemolymph of Tenebrio molitor,
  • Figure 6 is a graph of the phenoloxidase activity of the phenoloxidase composition isolated from the plasma of Tenebrio molitor depending on the concentration of ⁇ -1 ,3-glucan;
  • Figure 7 is a graph comparing phenoloxidase activity induced by ⁇ -1 ,3-
  • Figure 8 is a graph showing the phenoloxidase activity of the mixture of phenoloxidase composition isolated from the plasma of larvae of Holotrichia diomphalia and hemocyte lysate according to the reaction time in the presence
  • Figure 9 is a graph showing the result of ⁇ -1 ,3-glucan detection using the
  • Figure 10 is a graph showing the result of ⁇ -1 ,3-glucan detection using
  • Figure 11 is a graph showing the result of ⁇ -1 ,3-glucan detection using
  • Example 1 The present invention is further illustrated by way of the following Examples, but by no means limited thereto.
  • Example 1 The present invention is further illustrated by way of the following Examples, but by no means limited thereto.
  • glucan was identified in the presence or absence of Ca 2+ .
  • glucan were collected as secondarily purified phenoloxidase composition and it
  • the standard curve was obtained by measuring the phenoloxidase
  • the absorbance was measured at 520 nm after a one hour
  • ⁇ -1 ,3-glucan concentration range between 0 and 200 pg/ml.
  • lipopolysaccharide and peptidoglycan Lipopolysaccharide (Sigma Chemical Company) and peptidoglycan (Sigma) were each suspended in 50 mM of Tris buffer solution (pH 7.0). As substrates, peptidoglycan suspension was used as it is and lipopolysaccharide suspension was used after sonication for 2-3 min.
  • lipopolysaccharide at 200pg/ml, 20ng/ml, 20 ⁇ g/ml concentrations as
  • the color development reaction with 4-MC/4-MP solution was carried out by using phenoloxidase composition at 5 mM Ca 2+ .
  • the color was
  • lipopolysaccharide 200pg/ml ISI : A + lipopolysaccharide 20ng/ml, : A +
  • 0 A + peptidoglycan 200pg/ml
  • ESI A + peptidoglycan 20ng/ml
  • the phenoloxidase activity does not appear at high concentration, 20ug/ml of lipopolysaccharide and peptidoglycan even if the reaction time is increased. Therefore the phenoloxidase composition of the
  • present invention can detect ⁇ -1 , 3-glucan specifically.
  • the cells were sonicated and centrifuged for 5 min at 4 °C at 372 g to obtain 3 ml
  • phenoloxidase composition of partially purified plasma, hemocyte lysate and the phenoloxidase composition of primarily purified hemolymph of Example 1 were compared by using the same amount (400 ⁇ g) of proteins in the presence of
  • the phenoloxidase composition obtained from the hemolymph can detect ⁇ -1 ,3-
  • the hemocyte precipitate were obtained as plasma and hemocytes, respectively.
  • the hemocyte precipitate (5 ml) was sonicated after adding 5 ml of 50mM Tris buffer
  • the 4-MC/4-HP color development reaction was carried out for 30 min by setting
  • Figure 8 shows the ⁇ -1 , 3-glucan detection ability using the above
  • Blood was collected from 11 healthy adult males and females and 50 hospitalized cancer patients. After treating the collected blood with heparin, the
  • concentration of ⁇ -1 , 3-glucan is the value of average ⁇ SEM). In the latter, it is
  • 3-glucan as a cell wall component can be diagnosed at an early stage of the infection.
  • the mortality can then be lowered by administering appropriate antibiotics or antifungal drugs since it is

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)

Abstract

La présente invention concerne une composition qui permet de détecter une quantité infinitésimale de bêta-1,3-glucane dans un échantillon, un procédé de préparation de cette composition et une trousse de diagnostic utilisée pour détecter le bêta-1,3-glucane. La composition selon la présente invention présente une activité phénoloxydase par le bêta-1,3-glucane en présence d'ions calcium. Au moyen de la composition selon l'invention, un échantillon est obtenu à partir d'un prélèvement, la composition selon l'invention et des ions calcium sont ajoutés à l'échantillon et le bêta-1,3-glucane est détecté au moyen de la mesure de l'activité phénoloxydase.
EP01942566A 2000-01-20 2001-01-20 Composition de detection de beta-1,3-glucane, procede de preparation de cette derniere et trousse de diagnostic permettant de detecter le beta-1,3-glucane Ceased EP1274466A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
KR20000002542 2000-01-20
KR2000002542 2000-01-20
PCT/KR2001/000106 WO2001052905A1 (fr) 2000-01-20 2001-01-20 Composition de detection de beta-1,3-glucane, procede de preparation de cette derniere et trousse de diagnostic permettant de detecter le beta-1,3-glucane

Publications (2)

Publication Number Publication Date
EP1274466A1 true EP1274466A1 (fr) 2003-01-15
EP1274466A4 EP1274466A4 (fr) 2004-12-29

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EP01942566A Ceased EP1274466A4 (fr) 2000-01-20 2001-01-20 Composition de detection de beta-1,3-glucane, procede de preparation de cette derniere et trousse de diagnostic permettant de detecter le beta-1,3-glucane

Country Status (7)

Country Link
US (1) US6987002B2 (fr)
EP (1) EP1274466A4 (fr)
JP (1) JP3657913B2 (fr)
CN (1) CN1206365C (fr)
AU (1) AU2891401A (fr)
CA (1) CA2397495C (fr)
WO (1) WO2001052905A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002101083A1 (fr) * 2001-06-08 2002-12-19 Samyang Genex Corporation Composition pour detecter le peptidoglycane et kit de diagnostic pour detecter le peptidoglycane
DE602004022325D1 (de) * 2003-10-31 2009-09-10 Immunetics Inc Der kontamination von blutplättchen mit bakterien
US7598054B2 (en) 2003-10-31 2009-10-06 Immunetics, Inc. Rapid peptidoglycan-based assay for detection of bacterial contamination of platelets
US8450079B2 (en) 2003-10-31 2013-05-28 Immunetics, Inc. Method for detecting bacteria
WO2010107068A1 (fr) * 2009-03-17 2010-09-23 和光純薬工業株式会社 MÉTHODE DE MESURE DES β-GLUCANES ET PROTÉINE SE LIANT AUX β-GLUCANES POUR APPLICATION À LA PRÉSENTE MÉTHODE
CU24073B1 (es) 2011-12-27 2015-01-29 Ct De Investigación Y Desarrollo De Medicamentos Cidem COMPOSICIÓN A PARTIR DE EXTRACTO DE HEMOCITOS DE LANGOSTA PARA LA DETECCIÓN DE LIPOPOLISACÁRIDOS, PEPTIDOGLICANOS Y 1,3-ß-D-GLUCANOS
CN103789254A (zh) * 2012-10-26 2014-05-14 沈阳药科大学 昆虫、昆虫血淋巴及其组装活性物和应用
CN108663509A (zh) * 2017-06-16 2018-10-16 江苏诺鬲生物科技有限公司 一种标记真菌的免疫显色试剂

Citations (2)

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Publication number Priority date Publication date Assignee Title
EP0270039A2 (fr) * 1986-12-03 1988-06-08 Wako Pure Chemical Industries Ltd Réactifs pour la détermination de peptidoglycane et bêta-1,3-glucane
EP0757103A2 (fr) * 1995-07-31 1997-02-05 Wako Pure Chemical Industries Ltd Méthode pour la détection de microorganismes

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SE431228B (sv) 1981-12-17 1984-01-23 Kenneth Soderhell Sett att bestemma bakteriella endotoxin medelst profenoloxidashaltigt blodkroppslysat fran kreftdjur eller insekter och reagens herfor
JPH07114706B2 (ja) 1986-12-03 1995-12-13 和光純薬工業株式会社 新規測定試薬
JP2944709B2 (ja) * 1990-06-21 1999-09-06 生化学工業株式会社 （１→３）―β―Ｄ―グルカンの測定剤
DE69430038T2 (de) * 1993-11-18 2002-10-24 Wako Pure Chem Ind Ltd Verfahren zur Bestimmung der Aktivität des Prophenoloxidaseaktivierenden Enzyms und dessen Anwendung
JPH09131174A (ja) 1995-09-08 1997-05-20 Wako Pure Chem Ind Ltd 微生物検出用プレ−ト
EP0924220A3 (fr) * 1997-12-16 2000-04-26 Wako Pure Chemical Industries, Ltd. Inhibiteur de l'activation d'une protéine reconnaissant de beta-glucane
JPH11178599A (ja) 1997-12-22 1999-07-06 Wako Pure Chem Ind Ltd エンドトキシン又は／及びペプチドグリカン測定用試薬

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0270039A2 (fr) * 1986-12-03 1988-06-08 Wako Pure Chemical Industries Ltd Réactifs pour la détermination de peptidoglycane et bêta-1,3-glucane
EP0757103A2 (fr) * 1995-07-31 1997-02-05 Wako Pure Chemical Industries Ltd Méthode pour la détection de microorganismes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
KWON T H ET AL: "Purification and characterization of prophenoloxidase from the hemolymph of coleopteran insect, Holotrichia diomphalia larvae." MOLECULES AND CELLS. 28 FEB 1997, vol. 7, no. 1, 28 February 1997 (1997-02-28), pages 90-97, XP009039246 ISSN: 1016-8478 *
KWON TAE HYUK ET AL: "A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect, Holotrichia diomphalia larvae" EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 267, no. 20, October 2000 (2000-10), pages 6188-6196, XP002303959 ISSN: 0014-2956 *
LEE KWANG MOON ET AL: "Activated phenoloxidase from Tenebrio molitor larvae enhances the synthesis of melanin by using a vitellogenin-like protein in the presence of dopamine" EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 267, no. 12, June 2000 (2000-06), pages 3695-3703, XP002303957 ISSN: 0014-2956 *
LEE SO YOUNG ET AL: "In vitro activation of pro-phenol-oxidase by two kinds of pro-phenol-oxidase-activating factors isolated from hemolymph of coleopteran, Holotrichia diomphalia larvae" EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 254, no. 1, May 1998 (1998-05), pages 50-57, XP002303960 ISSN: 0014-2956 *
LEONARD C ET AL: "STUDIES ON PROPHENOLOXIDASE AND PROTEASE ACTIVITY OF BLABERUS-CRANIIFER HEMOCYTES" INSECT BIOCHEMISTRY, vol. 15, no. 6, 1985, pages 803-810, XP002303958 ISSN: 0020-1790 *
See also references of WO0152905A1 *

Also Published As

Publication number Publication date
CN1206365C (zh) 2005-06-15
AU2891401A (en) 2001-07-31
US20020197662A1 (en) 2002-12-26
CA2397495A1 (fr) 2001-07-26
CA2397495C (fr) 2010-03-16
EP1274466A4 (fr) 2004-12-29
JP2003520043A (ja) 2003-07-02
JP3657913B2 (ja) 2005-06-08
WO2001052905A1 (fr) 2001-07-26
US6987002B2 (en) 2006-01-17
CN1406139A (zh) 2003-03-26

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